WO2003092627A2 - Technique de criblage a haut debit pour detecter une sequence d'adn - Google Patents

Technique de criblage a haut debit pour detecter une sequence d'adn Download PDF

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WO2003092627A2
WO2003092627A2 PCT/US2003/014024 US0314024W WO03092627A2 WO 2003092627 A2 WO2003092627 A2 WO 2003092627A2 US 0314024 W US0314024 W US 0314024W WO 03092627 A2 WO03092627 A2 WO 03092627A2
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dna
container
nucleic acid
lysis buffer
samples
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PCT/US2003/014024
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WO2003092627A3 (fr
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Alex J. Harvey
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Avigenics, Inc.
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Priority to AU2003231309A priority Critical patent/AU2003231309B2/en
Priority to JP2004500812A priority patent/JP2005524397A/ja
Priority to EP03724449A priority patent/EP1503627A4/fr
Publication of WO2003092627A2 publication Critical patent/WO2003092627A2/fr
Publication of WO2003092627A3 publication Critical patent/WO2003092627A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates generally to a screening assay and, more specifically, to a high-throughput screening assay useful for detecting the presence of a foreign DNA sequence in a sample.
  • the present invention further includes a high throughput extraction method for extracting DNA from nucleated cells, particularly red blood cells.
  • the present invention provides a high throughput screening assay useful for detecting the presence of an exogenous DNA sequence in a sample.
  • the method of the present invention further includes a high throughput DNA extraction method useful for extracting DNA from avian blood for subsequent use in a screening assay as, for example, an assay to detect the insertion of foreign DNA in the genome of a recipient.
  • Transgenesis is the ability to introduce foreign or exogenous DNA into the genome of a recipient, as for example, into a sheep, a cow or even a chicken.
  • the ability to alter the genome of an animal immediately suggests a number of commercial applications, including the production of an animal able to express an exogenous protein in a form that is harvested easily.
  • the main obstacle to avian transgenesis is the low efficiency of introduction of foreign DNA into the chicken genome.
  • the insertion of foreign DNA into the chicken genome using procedures that have worked for other animals is a difficult task and attempts at such have been mostly unsuccessful, partly due to the unique physiology of the chicken (Love et al.. Transgenic birds by DNA microinjection, Biotechnology 12: 60- 63, 1994; Naito et al.. Introduction of exogenous DNA into somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova, Mol ReprodDev 37: 167-171, 1994).
  • Retroviral vectors are typically injected into the embryo of a freshly laid egg through a small window in the egg shell. Approximately 1% of the embryonic cells are transduced, such that one copy of the transgene is inserted into the cell's DNA.
  • sperm or oocytes carry the transgene.
  • at least 200 chicks have to be screened. It is often desirable to obtain several transgenic chicks because different chromosomal insertions can lead to different levels of transgene expression. Thus, it is necessary to breed and screen hundreds to thousands of chicks, necessitating a method for high throughput genetic screening for detecting the desired genetic sequence.
  • Random chromosomal insertion of transgenes via non-retroviral methods has become the mainstay of transgenics in some domesticated animals including pigs, sheep, goats and cows.
  • the primary method to introduce the transgene is the injection of linearized DNA containing the desired transgene into the pronucleus of a zygote. Up to 20% of G o offspring contain the transgene.
  • the relative high efficiency of transgenesis offsets the high technical costs incurred during the procedure.
  • Transgenes have been inserted into goats, for instance, that direct the expression of pharmaceuticals in mammary glands for subsequent secretion into milk (Ebert, et al., Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression, Biotechnology 9: 835- 8, 1991).
  • BDCs blastodermal cells
  • the embryonic blastoderm consists of 40,000 to 60,000 cells organized as a sheet (area pellucida) surrounded by the area opaca; it harbors presumptive primordial germ cells (PGCs) that have not yet differentiated into migrating PGCs.
  • PGCs primordial germ cells
  • Dispersed BDCs can be transfected with an appropriate transgene and introduced into the subgerminal cavity of y-irradiated, recipient stage X embryos. Irradiation may selectively destroy presumptive PGCs and retard recipient embryo growth allowing injected cells additional time to populate the recipient blastoderm.
  • mice ES cells The long- term culture of mouse ES cells allows the researcher to select for and expand colonies of cells transfected with the targeting vector that have the transgene inserted into the proper site. Similar to the use of the feather color alleles in chimeric birds, coat color of different breeds of mice are used to track the donor cells in offspring.
  • the difficulty in applying the mouse ES cell technology to other species is that it has been impossible to isolate ES cells of other species. While cells resembling ES cells have been isolated from goats and pigs and cultured in vitro, these cells are not able to contribute to recipient embryos after long-term culture.
  • Nuclear transfer technology offers an alternative to the use of ES cells and it is probable that gene targeting in animals will, in the future, be implemented via nuclear transfer. Presently, however, nuclear transfer is very inefficient and expensive, making its implementation a slow process.
  • stage X BDCs can be maintained in vitro at one division every 8 -10 hours for 4 - 8 days using culture conditions developed by the Ivarie laboratory (University of Georgia, Athens, GA) and AviGenics, Inc. (Athens, GA) (Speksnijder and Baugh, unpublished data).
  • the ability to propogate BDCs in vitro at this rate, while maintaining totipotency, will allow for the rapid expansion of cell colonies containing the desired genetic modification.
  • BDCs can only be cultured for 4 to 8 days before they lose the ability to contribute to germ tissues in the recipient embryo (Speksnijder and Baugh, unpublished data). Therefore, it is likely that BDCs carrying the desired genetic modification can only be enriched to perhaps 0.1 to 10% of the total number of donor cells. While sufficient to enable gene targeting, the rate of transmission of the desired genetic modification from chimeric founder animals (those that were directly derived from injection of donor BDCs into recipient embryos) to their offspring will be low. Hundreds to thousands of offspring will have to be screened, again necessitating a method for high throughput genetic screening for detecting a desired sequence.
  • DNA is extracted from a tissue sample (blood, skin, sperm) and is subjected to an assay that will detect the gene.
  • the method of choice was the Southern assay, which is extremely sensitive and reliable (Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J Mol Biol 98, 503-17, 1975).
  • the Southern assay is very labor intensive and time consuming.
  • the Southern assay was replaced by the polymerase chain reaction (PCR) method (Mullis et al., Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 51 (Pt 1): 263-73, 1986), which is a more sensitive and rapid assay.
  • PCR polymerase chain reaction
  • TAQMAN sequence detection system Applied Biosystems, Foster City, CA
  • TAQMAN sequence detection system Applied Biosystems, Foster City, CA
  • a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher fluorescent dye attached, anneals specifically between the forward and reverse primers.
  • the probe and primers are complementary to the sequence of the desired transgene.
  • the reporter dye When the probe is cleaved by the 5' nuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored during the PCR. Samples are analyzed in 96-well plates and, at the end of a run, it is obvious which samples contain the desired sequence.
  • Ramirez-Solis et al. devised an ingenious method in which ES cells are lysed in 96- well plates (Ramirez-Solis et al., Genomic DNA microextraction: a method to screen numerous samples. Anal Biochem 201: 331-5, 1992). Using the method of Ramirez- Solis, et al., DNA is precipitated such that it sticks to the bottom of the microtiter well without centrifugation.
  • Ramirez-Solis et al. attempted to isolate DNA from human blood samples using the above-described method, however the inefficiency of the procedure required processing a large volume of blood to obtain enough cells for efficient extraction. At least 0.3 ml, and most probably about 1.0 ml, of human blood is required per well to obtain enough DNA for efficient extraction, however the maximum capacity of each microtiter well is only about 0.25 ml. Thus, the method of Ramirez-Solis, et al. is not useful for the high throughput extraction of DNA from genomic blood.
  • Udy and Evans developed a 96-well plate method for DNA extraction from embryonic stem (ES) cells, similar to the method of Ramirez-Solis et al., but never applied their method to the extraction of DNA from blood (Udy and Evans, Microplate DNA preparation, PCR screening and cell freezing for gene targeting in embryonic stem cells, Biotechniques 17: 887-94, 1994).
  • the present invention recognizes and addresses the above noted deficiencies and drawbacks of the prior art.
  • the present invention provides a rapid method for extracting and preparing DNA for use in a subsequent high-throughput screening assay.
  • the method of the present invention is especially useful for extracting DNA from avian blood for use in a high throughput screening assay as, for example, an assay to detect the insertion of foreign DNA in the genome of a recipient.
  • the present invention is also directed to an assay useful for rapidly screening a large number of nucleated blood samples to detect a desired genetic sequence.
  • the nucleated blood samples may be avian blood such as, for example, from a chicken or turkey.
  • the genetic sequence may be an endogenous DNA gene or a foreign sequence such as, for example, a transgene, or alternately, the genetic sequence may be a plasmid.
  • a nucleic acid is isolated from a nucleated blood sample, particularly an avian blood sample, by placing the sample in a microtiter well, lysing the cells to lyse the plasma membrane, centrifuging the sample to recover a pellet, lysing the pellet for less than eight hours to release the DNA, precipitating the nucleic acid within the well of the microtiter plate such that the nucleic acid is attached to the well, removing any extraneous material from the well by washing, and subjecting the isolated nucleic acid to a screening assay to detect a desired genetic sequence.
  • lysis of the cell pellet is performed for between about one and about six hours to release the nucleic acid.
  • the present invention also provides a high throughput assay for detecting a desired sequence; the assay fiirther comprising a sequence tag that permits a desired genetic sequence to be detected at low copy numbers even in the presence of interfering genomic DNA.
  • the high-throughput assay provides a sequence tag which permits a target plasmid to be detected in the presence of chicken genomic DNA at a level of from about 5 to about 50 copies.
  • Figure 1 is a schematic illustrating the method of the present invention
  • Figure 2 is a photograph of an agarose gel.
  • DNA was extracted from blood obtained from White Leghorn chickens using either a conventional phenol-based method or the method of the present invention. After extraction, DNA samples were quantitated by absorbance at 260 nanometers and 1, 2 and 5 ⁇ g of each sample was separated on a 0.8% agarose gel. Samples extracted using the phenol-based method are shown in lanes marked as L, while lanes marked as H contain DNA samples extracted according to the method of the present invention.
  • Lane M contains a DNA standard with molecular sizes indicated as kilobase pairs
  • Figure 3 is a graph illustrating results of an experiment performed as described in Example 3, using the high throughput DNA extraction method of the present invention with an assay to detect the insertion of the chicken glyceraldehyde-3- phosphate dehydrogenase (GAPDH) gene.
  • Primers and a FAM/TAMRA-modified oligonucleotide probe complementary to the chicken GAPDH gene was used in a TAQMAN reaction to confirm the reliability of the high throughput DNA extraction method;
  • Figure 4 is a graph illustrating results of an experiment conducting high throughput screening of transgenic offspring according to the present invention.
  • DNA was extracted from 82 chicks that were bred from a male that was partially transgenic.
  • a TAQMAN reaction with primers and a TET/TAMRA-modified probe complementary to the bacterial neomycin resistance gene was used to detect the presence of the transgene.
  • Curves that did not demonstrate an increase in ⁇ Rn until after cycle 33 indicate that the respective chicks were not transgenic.
  • DNA extracted from a transgenic chick gave rise to amplification at cycle 18.
  • FIG. 5 is a schematic of a targeted gene showing the sequence tag.
  • the targeting vector is modified such that the sequence tag (Tag) is inserted at the 3' end of the polyadenylation signal sequence (pA).
  • the vector Upon introduction of the vector into the desired cells, the vector recombines with the target gene.
  • DNA is extracted from the cells and screened for those with a targeted gene using a PCR assay with primers NeoRev-1 and primer 2.
  • a TAQMAN probe (Neoprobe) can be added to the reaction if a realtime PCR reaction is to be run;
  • Figure 6 shows the nucleotide sequence of the sequence tag
  • Figure 7 is an agarose gel showing the results of an experiment using the high throughput assay and sequence tag of the present invention. Results showed detection of the targeted gene at copy number, even in the presence of 150 ng of chicken genomic DNA.
  • Each sample comprising 10 microliters of a TAQMAN reaction, was run on a 1% agarose gel and stained with ethidium bromide.
  • Lane one is one microgram of one kB DNA Ladder from Gibco-BRL.
  • Plasmid DNA is TTV-TTrev. The number of copies of plasmid in each reaction is indicated above each lane. The desired 941 bp product is indicated.
  • Figure 8 is a graph depicting real-time PCR detection of a targeted gene using the high throughput assay of the present invention. The reactions were conducted as specified in Figure 7 above.
  • a rapid method for extracting and preparing DNA for use in the high-throughput screening assay is provided.
  • the method of the present invention is especially useful for extracting DNA from nucleated blood for use in a high throughput screening assay including, but not limited to, a polymerase chain reaction (PCR), ligase chain reaction (LCR), or other conventional DNA detection assay for the detection of genetic markers or foreign DNA in the genome of a recipient.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • a high throughput method for extracting DNA from multiple samples of chicken blood is disclosed, as for example, from White Leghorn and Barred Rock chicks and fully mature birds.
  • the method of the present invention can be used for the high throughput extraction of a DNA from any nucleated blood cell including, but not limited to, avian, fish, reptile and amphibian nucleated blood cells.
  • the present invention provides a high throughput method for extracting DNA from multiple blood samples containing nucleated blood cells without requiring the repositioning of the DNA into separate tubes or vessels during the extraction procedure.
  • a nucleic acid such as DNA is extracted from a nucleated blood sample, particularly an avian blood sample, by placing the sample in a microtiter well, lysing the cells to release the DNA, precipitating the nucleic acid within the well of the microtiter plate such that the nucleic acid is attached to the well, removing any extraneous material from the well by washing, and subjecting the isolated nucleic acid to an assay.
  • the present invention further provides a sequence tag for use in a high throughput assay to permit detection of the desired genetic sequence at low copy numbers.
  • the sequence tag is used in the high throughput assay of the present invention to allow a plasmid to be detected at a level of from about 5 to about 50 copies in the presence of chicken genomic DNA.
  • a high throughput DNA extraction method adapted for use with blood from species other than avian.
  • an alternate embodiment of the present invention provides a DNA extraction method that uses mammalian red blood cells in the high throughput assay of the present invention.
  • mammalian blood is enriched for those RBCs that are nucleated, as with cell sorting, centrifugation, or the administration of a hemopoietic agent, such that a sufficient amount of nucleated cells can be transferred to each well of a microtiter plate in a volume of 250 ⁇ l or less.
  • DNA or other nucleic acid is exfracted from nucleated cells other than red blood cells.
  • white blood cells including granulocytes, neutrophils and mast cells, can be used in the high throughput assay of the present invention.
  • Example 1 DNA Extraction Method Briefly, the protocol for DNA extraction from avian blood according to the present invention is as follows:
  • lysis buffer LB1 containing 0.32 M sucrose, lOmM Tris-Cl, 5 mM MgC12, and 1% Triton X-100, at pH 7.5
  • Duplicate plates were set up for each set of 96 chicks. The 96-well plates were kept on ice until step C below.
  • one drop (about 8 microliter or '/4 th of the capillary) of blood was transferred into one well and its duplicate, each containing LB1. Following transfer, the blood and LB1 were mixed in each well using the capillary tube. If chicks older than 10 days are used as blood donors, a 25G needle and 1 cc syringe primed with 0.05 ml of heparin can be used to collect blood. Transfer one drop (about 8 ⁇ l) into each well.
  • lysis solution can hold only so much blood, otherwise the quality of the DNA will significantly decrease. Add enough blood such that the lysis solution is light to medium red. If significant clotting occurs, the cell pellet is lost during subsequent steps, or the DNA appears yellow or brown after resuspension, it is likely that too much blood was added to LB 1.
  • Each microtiter plate was centrifuged at about 960 g (about 2000 rpm in a tabletop centrifuge) for 7 minutes to pellet nuclei.
  • lysis buffer 2 (LB2 containing 10 mM Tris-Cl, 10 mM NaCl, lOmM EDTA, and 1 mg/ml proteinase K at pH 8.0) was added to each well, and the plates incubated for between one and eight hours at 56 - 65°C. Incubation time can vary, but for optimal results, incubation with the second lysis buffer should be about 2 - 6 hours. Around 8 hours of incubation, the samples become unuseable due to DNA degradation.
  • the supernatent was then removed by carefully inverting the plate and pouring the supernatent into a large beaker.
  • a schematic is provided to illustrate the steps of the DNA extraction method according to the present invention. As illustrated in the schematic, 8 to 12 ⁇ l of avian blood is added to lysis buffer 1 (LB1) in each well of a
  • 96-well plate After lysis of the red blood cell plasma membrane occurs, the nuclei are spun down and the supernatents containing cytoplasmic proteins are removed. A proteinase K solution is added such that the bed of nuclei is not disturbed. After lysis of the nuclei, a solution of ethanol and NaCl is gently added. The chromosomal DNA precipitates and forms a dense white mat that adheres tightly to the bottom of the well.
  • the DNA mat can be easily washed with 70% ethanol several times without centrifugation. The solutions are simply poured off by hand between each wash.
  • the plate is inverted onto some paper towels, dried and water is added to each well to resuspend the DNA.
  • the amount of DNA present in each well does not need to be quantitated. Rather, after the last 70% ethanol wash and before drying, a visual inspection of the plate will indicate which wells do not have an adequate amount of DNA. A well containing an adequate amount of DNA will have a dense white mat of DNA at its bottom, which is easily visualized if the plate is held up against a black background.
  • Example 2 Average DNA Yield Using High Throughput DNA Extraction Three separate DNA extraction experiments were conducted using blood samples obtained from White Leghorn chickens as described in Example 1 above. To quantify yield following high throughput extraction, 2ul of DNA was added to 5 ul of Picogreen (Molecular Probes, Eugene, OR) in 1.0 ml of TE buffer (containing 0.1 M Tris-base, and 0.005 M EDTA at pH 7.5). Samples were read on a Turner Designs TD-700 Fluorometer using CsCl-banded plasmid DNA quanitated by absorbance at A 260 as a standard
  • FIG 2 a photograph of an agarose gel is presented which compares DNA extracted according to the method of the present invention with that obtained using a conventional phenol-based method (see, for example, the standard phenol extraction protocol provided in "Molecular Cloning: A Laboratory Manual,” 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Press, 1989 and Methods in Plant Molecular Biology: A Laboratory Course Manual, P. Maliga et al, eds., Cold Spring Harbor Press, 1994).
  • Blood obtained from White Leghorn chickens was extracted according to either the high throughput method of the present invention, as described in Example 1, or a conventional phenol based method.
  • DNA samples were quantitated by absorbance at 260 nanometers, loaded onto an 0.8% agarose gel (at 1, 2 and 5 ⁇ g concentrations of DNA) and subjected to electrophoresis using a conventional protocol.
  • the gel was visualized using an ethidium bromide stain to compare the quality of the DNA exfracted according to the present invention (lanes marked as H) with that exfracted using a conventional phenol-based technique (lanes marked as L).
  • Lane M contains a DNA standard with molecular sizes indicated.
  • the quality of the DNA extracted using the high throughput method of the present invention is comparable to that extracted with the conventional technique.
  • Example 3 Identification of a GPDH Transgene in the Chicken Genome Using the High Throughput Assay
  • a primer/probe set complementary to the chicken glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) was designed and made commercially.
  • the primers were made at Gibco BRL (Gaithersburg, MD) and the probe was synthesized by Operon Technologies (Alameda, CA).
  • the primers used were designed as follows: chGAPDH-l: 5'-TCCCAGATTTGGCCGTATTG-3' (SEQ ID NO: 1) and chGAPDH-2: 5'-CCACTTGGACTTTGCCAGAGA-3' (SEQ ID NO: 2).
  • the sequence of the chGAPDH probe was 5'-CCGCCTGGTCACCAGGGCTG-3' (SEQ ID NO: 3).
  • the chGAPDH probe was labeled with FAM (6-carboxyfluorescin) at the 5' end and TAMRA (N,N,N',N'-teframethyl-6-carboxyrhodamine) at the 3' end.
  • the TAQMAN assay measures the increase of relative fluorescence due to hybridization of the chGPDH probe to the PCR product and the resulting endonucleolytic cleavage of the probe. The cleavage releases the FAM molecule from the probe so that its fluorescence is no longer quenched by TAMRA.
  • TAQMAN reactions were carried out in 50 ul volumes by adding 100 to 300 ng of DNA, extracted from blood obtained from randomly-selected White Leghorn chicks according to the method of the present invention described in Example 1 above.
  • 0.75X PCR Buffer Perkin-Elmer, Foster City, CA
  • 0.25X TAQMAN buffer Perkin-Elmer
  • 2.5 mM MgC12 5% DMSO
  • 125 ⁇ M dATP 125 ⁇ M dCTP
  • 125 ⁇ M dGTP 250 ⁇ M UTP
  • 0.9 ⁇ M forward primer, 0.9 ⁇ M reverse primer 40 nM chGAPDH probe
  • 0.05 U/ ⁇ l AmpliTaq Gold DNA Polymerase Perkin-Elmer
  • 0.004U/ ⁇ l and AmpErase UNG Perkin-Elmer was added according to the manufacturer's recommendations.
  • Reactions were analyzed on a Perkin-Elmer Applied Biosystems Sequence Detector Model 7700 using the following conditions: 50° C for 2 minutes, 95° C for 10 minutes, followed by 40 or 50 cycles of 95° C for 15 seconds and 60° C for 1 minute.
  • results of the TAQMAN reaction were visualized as an increase in the fluorescence (eRn) during each cycle of the PCR reaction.
  • An increase in eRn at an earlier cycle indicates the presence of more copies of that particular sequence, whereas an increase in eRn at a later cycle indicates that fewer copies of the sequence are present.
  • TAQMAN data can determine the presence of a specific sequence and the relative quantity of that sequence.
  • FIG. 3 depicts the results of the TAQMAN amplification assay measuring fluorescence at each cycle of the PCR reaction. The cycle number is shown on the x- axis (only cycles 18-50 are shown).
  • ⁇ Rn is the increase of relative fluorescence due to hybridization of the chGAPDH probe to the PCR product and the resulting endonucleolytic cleavage of the probe.
  • the three control samples (blanks) produced overlapping curves that show no increase in ⁇ Rn, while the DNA samples obtained from all 21 White Leghorn chicks gave rise to very similar amplification plots showing hybridization of the probe to the chGAPDFI gene.
  • the sequences from which to choose the primers is limited to specific areas of the targeting vector and the target gene.
  • the primer specific for the targeting vector should reside within the 3' untranslated region (UTR) of the selection cassette.
  • UTR 3' untranslated region
  • most 3' UTRs are very short, limiting the choice of potential primer binding sites.
  • the primer binding site should reside relatively close to the 3' end of the 3' UTR to keep the length of the PCR product as short as possible. The longer the PCR product, the more inefficient the PCR reaction.
  • NeoRevl SEQ ID NO. 6
  • results using the sequence tag with a template in the high throughput assay of the present invention show that the template can be detected in extremely low copy numbers (5 - 20 copies) even in the presence of genomic DNA (100 ng of chicken DNA).
  • the NeoRevl sequence tag can be used in combination with almost any primer that anneals to a site downstream of NeoRevl and primes DNA synthesis in the opposite direction.
  • a 62 bp sequence from the Neomycin resistance gene having the sequence GTG CCC AGT CAT AGC CGA ATA GCC TCT CCA CCC AAG CGG CCG GAG AAC CTG CGT GCA ATC CA (SEQ ID NO.: 5), was cloned into the bovine growth hormone 3' untranslated region (UTR) or polyadenylation sequence such that the new sequence resides just downstream of the UTR (see Figure 6). This positions a binding site for the primer NeoRevl (SEQ ID NO.: 6) that will prime DNA synthesis away from the UTR using a PCR reaction.
  • the PCR reactions are relatively insensitive to the type of polymerase used or the magnesium concentration, an indication of the robustness of the reaction.
  • the inserted sequence contains a binding site for a neomycin probe (Neoprobe; SEQ ID NO.: 7) that can be used in a variety of real-time PCR reactions, including TAQMAN (Perkin Elmer), allowing high tliroughput detection of a gene targeting event.
  • the inserted sequence contains a second primer binding site (NeoForl; SEQ ID NO.: 4) which primes synthesis in the direction opposite to that of NeoRevl.
  • the combination of these two primers and the probe enables detection of this sequence, regardless of the sequence context, in an efficient and high throughput manner. Because the amplicon is short (62 bp), amplification is highly efficient.
  • This primer set can be used in a quantitative PCR reaction (realtime or gel-based) to accurately determine the copy number of the transgene. This would be useful, for example, if a transgene has integrated randomly because, in many cases of random insertion, multiple copies of the transgene inserts. Thus, one is required to dete ⁇ nine the copy number of the transgene.
  • a second example in which copy number must be determined occurs when the animals are bred to be homozygous for the transgene. In this case, desired animals have twice as many copies of the fransgene as their parents orhemizygous (single copy) siblings.
  • a targeting vector was constructed by subcloning of the 62 bp sequence (SEQ ID NO.: 5) shown in Fig. 6 into a restriction site at the 3' end of the polyadenylation signal.
  • a 62 bp product was produced by PCR by using the neomycin resistance gene (E.coli Transposon Tn5) as the template and using the following primers:
  • NeoForl 5'-TGGATTGCACGCAGGTTCT-3' (SEQ ID NO.: 4), and NeoRevl: 5'-GTGCCCAGTCATAGCCGAAT-3' (SEQ ID NO.: 6).
  • the primers were kinased with T4 DNA Kinase and ATP prior to PCR.
  • the vector was cut with a restriction site that produced a blunt end and ligated to the PCR product.
  • a subclone was selected in which the PCR product had inserted in the reverse orientation such that the NeoRevl primer primed DNA synthesis away from the polyadenylation signal, as shown in Figure 5.
  • this vector is referred to as Targeting Vector-Transgene Tag-rev or TV-TTrev.
  • the 3' flank of the targeting vector which is homologous to a region of the chicken ovalbumin gene, was replaced by a longer segment of the same region of the gene.
  • This vector is referred to as Targeting Test Vector-Transgene Tag-rev or TTV-TTrev.
  • TTV-TTrev A clone in which the PCR product was in the forward orientation was also selected.
  • the NeoForl sequence tag primes DNA synthesis away from the polyadenylation signal.
  • the analogous test vector is referred to as Targeting Test Vector-Transgene Tag-for or TTV-TTfor. When this vector is used, the NeoForl sequence tag would be used to prime DNA synthesis.
  • Results comparing a high throughput detection assay for TTV-TTfor using the NeoForl sequence tag and OV18rev primer (SEQ ID NO.: 8; 5'-CAA TAG AAG ATT TAT ACT TGT TCT GTC TGT TT) with an assay detecting TTV-TTrev with NeoRevl and OVl ⁇ rev show the NeoForl sequence tag and OV18rev assay has a much lower sensitivity (10-100 fold) than that of the TTV-TTrev and primers NeoRevl and OV18rev.
  • NeoRevl sequence tag The sensitivity of detection using the NeoRevl sequence tag was tested as follows: TAQMAN reactions were carried out in 20 ul volumes and all reactions had 150 ng of White Leghorn DNA, extracted from blood obtained from randomly- selected chicks according to the method of the present invention described in Example 1 above.
  • AmpliTaq Gold DNA Polymerase was replaced with Promega Taq DNA polymerase (Promega, Madison, WI). Additionally the Perkin-Elmer dNTPs/UTP mixture can be substituted with dNTPs from Roche (catalog number 1969064, Indianapolis, IN). Reactions containing AmpliTaq Gold DNA Polymerase were analyzed on a Perkin-Elmer Applied Biosystems Sequence Detector Model 7700 using the following conditions: 50° C for 2 minutes, 95° C for 10 minutes, followed by 40 or 50 cycles of 95° C for 20 seconds and 62.8°C for 2 minutes, 30 seconds. The following conditions were used when Promega Taq DNA polymerase was in the reaction mixture: 94° C for 2 minutes, followed by 40 or 50 cycles of 94° C for 20 seconds and 62.8°C for 2 minutes, 30 seconds.
  • Figures 7 and 8 show the results of PCR experiments using the NeoRev-1 sequence tag (SEQ ID NO.: 6) as the forward primer and OVl ⁇ rev (SEQ ID NO.: 8) as .the reverse primer.
  • the expected 941 bp band is detectable in as low as 5 copies of plasmid DNA.
  • Figure 8 shows the results from a real-time PCR detection experiment using the sequence tag in the presence of 150 ng of chicken DNA. Results confirm the detection of the desired gene sequence at a 5 copy level.
  • Example 5 Detection of a Neomycin Resistance Gene in the Chicken Genome Using the High Throughput DNA Extraction Method
  • White Leghorn embryos were transduced with a retroviral vector containing the bacterial neomycm resistance gene (NeoR). Because of the inefficiency of fransduction, even in the best cases less than 1% of the embryonic cells, including those that give rise to germ tissues, carry a copy of the transgene. Males that arose from the transductions were bred to non-transgenic White Leghorn hens. The resulting chicks were hatched and DNA was extracted in duplicate via the high throughput DNA extraction method described in Example 1 above.
  • neomycin resistance gene was performed using the TAQMAN assay described in Example 3 above, except that the sequence of the primers used was as follows: NeoForl: 5'-TGGATTGCACGCAGGTTCT-3' (SEQ ID NO.: 4) and NeoRevl: 5'-GTGCCCAGTCATAGCCGAAT-3' (SEQ ID NO.: 6).
  • the sequence of the TAQMAN probe (Neoprobe), designed to be complementary to the bacterial neomycin resistance gene was 5 '-CCTCTCCACCCAAGCGGCCG-3 ' (SEQ ID NO.: 7).
  • Neoprobe was labeled with TET (tefrachloro-6-carboxy-fluorescein) or FAM (6-carboxyfluorescin) at the 5' end and TAMRA (N,N,N',N'-tetramethyl-6- carboxyrhodamine) at the 3' end. Reactions were carried out as described in Example 3 above.
  • Figure 4 shows the results of the neomycin detection assay. As can be seen in
  • this DNA extraction method can be used to facilitate the isolation of founder transgenic chicks, but also the method can be used to facilitate the propagation of those chicks into production flocks. Unless birds that are both homozygous for the desired transgene are mated to each other, only a percentage (50-75%>) of offspring from a transgenic founder will carry the transgene, necessitating the screening of thousands of chicks for the desired fransgene.
  • the method of the present invention also provides a significant impact for the screening of genetic markers that are associated with wanted or unwanted traits. Once identified, these traits can be enriched or selected against to produce genetically superior offspring using DNA extracted according to the present invention coupled with a screening assay.

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Abstract

La modification ou la sélection génétique d'espèces aviaires nécessite l'analyse génétique de grands nombres d'oiseaux afin de rechercher des séquences voulues. Généralement, l'ADN est extrait sur une base individuelle à partir d'échantillons prélevés sur les oiseaux. Dans les procédés actuels d'extraction d'ADN, l'ADN est extrait du sang ou d'autres tissus par la mise en oeuvre de procédures longues et laborieuses. L'invention concerne une technique de criblage à haut débit visant à détecter une séquence génétique dans de multiples échantillons. Cette technique comprend de plus un procédé d'extraction d'ADN qui permet d'extraire rapidement l'ADN de multiples échantillons aviaires, p. ex. globules rouges. Ce procédé d'extraction est extrêmement fiable et ne requiert pas de quantification de chaque échantillon après extraction. L'ADN extrait peut être utilisé dans diverses techniques génétiques, y compris une technique de criblage à haut débit, pour identifier l'insertion d'un transgène. L'invention est particulièrement utile pour extraire l'ADN de globules rouges nucléés. Par conséquent, ledit procédé peut être appliqué à l'analyse génétique d'espèces aviaires, de poissons, de reptiles et d'amphibiens.
PCT/US2003/014024 2002-05-02 2003-05-02 Technique de criblage a haut debit pour detecter une sequence d'adn WO2003092627A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690805A (zh) * 2011-12-01 2012-09-26 河南科技大学 一种快速提取禽类血液基因组dna的方法
CN103512789A (zh) * 2012-06-26 2014-01-15 李翰卿 一种提取样本中被分析物质的试剂以及提取的方法

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Publication number Priority date Publication date Assignee Title
US20100041039A1 (en) * 2000-01-15 2010-02-18 Synageva Biopharma Corp. Analysis of nucleic acid obtained from nucleated red blood cells
CN102827831A (zh) * 2012-08-31 2012-12-19 武汉百泰基因工程有限公司 一种核酸释放试剂及核酸释放方法
ES2882656T3 (es) * 2014-11-18 2021-12-02 Vertex Pharma Proceso para realizar pruebas de alto rendimiento de cromatografía líquida de alta resolución

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6423488B1 (en) * 2000-01-15 2002-07-23 Avigenics, Inc High throughput screening assay for detecting a DNA sequence

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2274793A1 (fr) * 1996-12-11 1998-06-18 Margaret Raybuck Lyse selective de cellules
FR2782324B1 (fr) * 1998-08-13 2002-12-06 Bruno Thuilliez Procede pour purifier et valoriser les acides desoxyribonucleiques ou adn contenus dans du sang de volaille

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6423488B1 (en) * 2000-01-15 2002-07-23 Avigenics, Inc High throughput screening assay for detecting a DNA sequence

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BERCOVICH ET AL: 'Improved protocol for using avian red blood cells as substrates for the polymerase chain reaction' BIOTECHNIQUES vol. 26, June 1999, pages 1080 - 1082, XP002978550 *
HOLLAND ET AL: 'Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of thermus aquaticus DNA polymerase' PNAS vol. 88, August 1991, pages 7276 - 7280, XP002930385 *
MEDRANO ET AL: 'DNA extraction from nucleated red blood cells' BIOTECHNIQUES vol. 8, no. 1, January 1990, page 43, XP002978575 *
PETITTE ET AL: 'Isolation of genomic DNA from avian whole blood' BIOTECHNIQUES vol. 17, no. 4, 14 July 1994, pages 664 - 666, XP002978551 *
RAMIREZ-SOLIS ET AL: 'Genomic DNA microextraction: a method to screen numerous samples' ANALYTICAL BIOCHEMISTRY vol. 201, January 1992, pages 331 - 335, XP000257967 *
SAMBROOK ET AL: 'Molecular cloning: a laboratory manual', 1993, COLD SPRING HARBOR LABORATORY PRESS pages B20 - B16 *
See also references of EP1503627A2 *
THORAVAL ET AL: 'Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors' TRANSGENIC RESEARCH vol. 4, January 1994, pages 369 - 377, XP002095225 *
UDY ET AL: 'Microplate DNA preparation, PCR screening and cell freezing for gene targeting in embryonic stem cells' BIOTECHNIQUES vol. 17, no. 5, 16 August 1994, pages 887 - 894, XP001194429 *
WITTWER ET AL: 'Continuous fluorescence monitoring of rapid cycle DNA amplification' BIOTECHNIQUES vol. 22, January 1997, pages 130 - 138, XP000683698 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690805A (zh) * 2011-12-01 2012-09-26 河南科技大学 一种快速提取禽类血液基因组dna的方法
CN102690805B (zh) * 2011-12-01 2014-12-24 河南科技大学 一种快速提取禽类血液基因组dna的方法
CN103512789A (zh) * 2012-06-26 2014-01-15 李翰卿 一种提取样本中被分析物质的试剂以及提取的方法

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